Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and/or combined with other pieces of prior art by a skilled person in the art.
Nontyphoidal Salmonella is the largest foodborne-disease burden in the United States, causing the most infections, hospitalizations and deaths, with 1.03 million illnesses reported annually. The economic burden associated with the disease is staggering, with the medical costs alone reaching more than $11 billion per year and substantial additional costs incurred by the food industry (recalls, litigation, reduced consumer confidence) and by state, local and federal public health agencies in response to NTS outbreaks. Globally, nontyphoidal Salmonella is estimated at 93.8 million cases and 155,000 deaths annually and has emerged as the leading cause of bacteremia in sub-Saharan Africa, where its fatality rate reaches up to 25%.
The health and economic burden associated with Salmonella is poised to worsen as the prolonged administration of antibiotics has resulted in the emergence of multidrug-resistant strains that have disseminated worldwide; e.g., S. Typhimurium DT104 has caused several food-borne disease outbreaks over the last two decades and is resistant to four of the five most commonly used antibiotics in veterinary medicine (tetracycline, β-lactams, aminoglycosides, and sulfonamides). These multidrug-resistant strains are oftentimes associated with more hospitalizations and bacteremia, and their maintenance in nature can occur at very low antibiotic concentrations that are commonly found in the environment including ground water. Further, a new class of carbapenem-resistant Enterobacteriaceae that are resistant to β-lactams, fluoroquinolones, and aminoglycosides was isolated from a patient in 2009, and such resistance has now shown widespread distribution among Gram-negative pathogens including Salmonella. Additionally, ‘hypervirulent’ Salmonella have been recently isolated (2012) from natural microbial populations derived from livestock. These hypervirulent strains are 100-times more virulent then most clinical isolates, are more capable of killing vaccinated animals, and are not detectable under standard laboratory test conditions due to rapid switching to a less-virulent state ex vivo. Together, these findings support the view that the Salmonella disease burden is poised to worsen with the potential emergence of more virulent multidrug-resistant strains that are difficult to control with currently available antibiotics.
Salmonella enterica is acquired via the fecal-oral route and is comprised of six subspecies that are subdivided into more than 2500 serovars (serological variants) based on carbohydrate, lipopolysaccharide (LPS), and flagellar composition, with subspecies enterica containing more than 99% of human pathogenic isolates. S. enterica infection can result in any of four distinct disease syndromes: enterocolitis/diarrhea, bacteremia, enteric (typhoid) fever and chronic asymptomatic carriage. Many serovars infect both humans and animals, and disease severity is a function of the serovar, strain virulence and host susceptibility.
Salmonella control efforts in livestock continue to be problematic for the following reasons: 1) most livestock infections are subclinical; 2) disease outbreaks are sporadic and frequently caused by specific serotypes although many serotypes are endemic to livestock production systems; 3) environmental persistence provides an ongoing reservoir for livestock infection; 4) the recent emergence of strain variants that are more virulent and can kill vaccinated animals; 5) some strains derived from human salmonellosis patients are distinct from those of animal origin; and 6) management and environmental events can increase pathogen exposure and/or compromise host immunity.
Vaccination represents a sustainable approach to any food safety plan, reducing pathogen exposure at the outset of the food production chain [1]. However, the immunity conferred by conventional vaccines is restricted to a narrow range of closely-related strains, and on-farm control requires the development of vaccines that elicit protection against many pathogenic serotypes [1]. Recent advancements have resulted in the development of modified live Salmonella cross-protective vaccines, many of which contain mutations in global regulatory networks that favor antigen production; and that are also suitable for the expression of heterologous antigens [2-6]. The molecular basis of cross-protective vaccine efficacy is not entirely clear. Relevant parameters might include: the expression of multiple antigens shared among pathogenic serotypes; diminished vaccine-induced immunosuppression; targeted removal of immunodominant antigens to expose cross-protective epitopes; type III secretion of recombinant antigens; and/or delayed vaccine attenuation for enhanced stimulation of immune responses (reviewed in [1, 3, 7, 8]).
Modified live attenuated S. enterica serovar Typhimurium that harbor loss of function mutations in genes may be useful for providing protection against a diversity of salmonella. The number of loci that might be considered for providing an applicable loss of function mutation is large, as is the number of applicable mutations at each locus. Some examples of loci for providing loss of function include loci involved in adherence, invasion, and intra- and extracellular survival of the bacteria (including many genes encoding proteins involved in metabolic processes). Some mutations of the gene encoding the DNA adenine methylase (dam) are capable of eliciting protection against a diversity of salmonellae. These appear to be well tolerated when applied as modified live vaccines in mice [2, 9], poultry [10, 11], sheep [12] and calves [13-15]. Induction of immunity is rapid and the vaccine can be administered with delivery via drinking water for low cost and low-stress immunization of livestock populations [12, 16].
The commercial success of any vaccine is dependent on the therapeutic index, the ratio of safety/toxicity, and safety is of particular concern for modified live vaccines that have the potential to revert to heightened virulence. Generally, a vaccine should satisfy 4 safety categories to be considered as a candidate for commercial use in a livestock production system. The relevant safety phenotypes are as follows: reduced i) vaccine shedding; ii) challenge strain shedding; iii) persistence in systemic tissues (liver/spleen); and iv) persistence in the environment.
It is understood that in providing an attenuated strain containing loss of function mutations, it is important that the improved safety profile arising from the relevant mutations does not decrease the efficacy of the vaccine in terms of the protection that it provides. Ultimately what one is looking for is a mutation that does not decrease the persistence of low level infection in the immunized individual and that does not increase the persistence of the immunogen in the environment when the pathogen is shed from the immunized animal and released to the environment.
It is difficult to predict which loss of function mutations are more useful for attenuation than others, particularly given that the data on potency and reversion to pathogenicity relevant to each locus and mutation arises from different laboratory systems.
A further complication is that it is desirable to select more than one locus for mutation, so as to prevent reversion to a pathogenic phenotype should at least one loss of function mutation be lost. Such an approach requires one to combine at least 2 loci from a large list of candidate loci and yet with limited guidance as to whether a particular combination is likely to increase or decrease the likelihood of reversion, or likely to increase or decrease the potency of the resulting attenuated vaccine.
There is a need for an attenuated live vaccine for protection against Salmonella infection.
There is also a need for an attenuated live vaccine for protection against Salmonella infection that has an improved safety phenotype.
There is a need for an attenuated live vaccine that has limited or no propensity for shedding from an animal, that has minimal persistence in the environment when shed from an animal, and that retains an acceptable level of persistence in an animal to invoke immunity.